in vitro antiviral effect of germacrone on feline calicivirus · received: 13 august 2015/accepted:...
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ORIGINAL ARTICLE
In vitro antiviral effect of germacrone on feline calicivirus
Hongxia Wu1 • Yongxiang Liu1 • Shaopo Zu1,2 • Xue Sun1,2 • Chunguo Liu1 •
Dafei Liu1 • Xiaozhan Zhang1 • Jin Tian1 • Liandong Qu1
Received: 13 August 2015 / Accepted: 10 March 2016 / Published online: 21 March 2016
� Springer-Verlag Wien 2016
Abstract Feline calicivirus (FCV) often causes respira-
tory tract and oral disease in cats and is a highly contagious
virus. Widespread vaccination does not prevent the spread
of FCV. Furthermore, the low fidelity of the RNA-depen-
dent RNA polymerase of FCV leads to the emergence of
new variants, some of which show increased virulence.
Currently, few effective anti-FCV drugs are available.
Here, we found that germacrone, one of the main con-
stituents of volatile oil from rhizoma curcuma, was able to
effectively reduce the growth of FCV strain F9 in vitro.
This compound exhibited a strong anti-FCV effect mainly
in the early phase of the viral life cycle. The antiviral effect
depended on the concentration of the drug. In addition,
germacrone treatment had a significant inhibitory effect
against two other reference strains, 2280 and Bolin, and
resulted in a significant reduction in the replication of
strains WZ-1 and HRB-SS, which were recently isolated in
China. This is the first report of antiviral effects of ger-
macrone against a calicivirus, and extensive in vivo
research is needed to evaluate this drug as an antiviral
therapeutic agent for FCV.
Introduction
Caliciviruses are non-enveloped, small, positive-strand
RNA viruses that are divided into five genera: Norovirus,
Lagovirus, Sapovirus, Vesivirus, and Nebovirus [1]. The
absence of an animal cell model for most caliciviruses has
restricted our understanding of their biology. In particular,
infection with human noroviruses (HuNoVs) has caused
viral epidemic gastroenteritis globally in people of all ages
[38]. Feline calicivirus (FCV) and murine norovirus have
been used widely as model systems [39], and this has
contributed to our understanding of HuNoVs. FCV belongs
to the genus Vesivirus and is a highly contagious pathogen
that is widely distributed in the feline population [41].
Previous FCV isolates mainly induced upper respiratory
tract disease and oral ulceration [22, 23, 31, 32] with a low
mortality rate (*2 %) [2, 40]. However, several outbreaks
with high mortality (up to 60 %) have been reported in
recent years [24].
The gene encoding the capsid protein of FCV contains a
variable region [33], and the evolutionary rate in this
region ranges from 1.3 9 10-2 to 2.6 9 10-2 substitu-
tions/nucleotide/year [4]. FCV has the highest evolutionary
rate among viruses [4]. Although there is only one serotype
of FCV, the variability of its main antigen VP1 often
contributes to a lack of efficacy of vaccination, and wide-
spread vaccination does not prevent the spread of FCV
[19]. In natural infections, cats that have recovered fully
from an FCV infection are not protected against infection
with other FCV strains [21]. In addition, FCV variants can
H. Wu and Y. Liu are co-first authors.
& Jin Tian
& Liandong Qu
Xiaozhan Zhang
1 State Key Laboratory of Veterinary Biotechnology, Harbin
Veterinary Research Institute, Chinese Academy of
Agricultural Sciences, 427 Maduan Street, Nangang District,
Harbin 150001, People’s Republic of China
2 College of Veterinary Medicine, Northeast Agricultural
University, Harbin, People’s Republic of China
123
Arch Virol (2016) 161:1559–1567
DOI 10.1007/s00705-016-2825-8
inhibit or evade the host immune response and persist in
cats [4]. After recovery from FCV infection, the clinical
symptoms disappear, but many cats continue shedding
virus for more than 30 days or even several years [32]. Our
previous study found that FCV strain 2280 cannot induce
IFN-b expression in vitro [34], which may be a key factor
for survival of FCV in cats. Failure to efficiently control
FCV infection leads to a high prevalence of FCV. FCV
vaccines reduce the duration and severity of clinical signs
but do not inhibit virus shedding or infection [25].
Since FCV vaccines do not provide a complete protec-
tion, it is urgent to develop an effective and safe antiviral
drug for monotherapy or combination treatment. Produc-
tion of type I interferons (IFNs), which are involved in
antiviral responses and have broad-spectrum antiviral
activities, is triggered by the presence of viral dsRNA or
by-products of viral replication [10]. However, many
viruses have evolved multiple strategies to evade or inhibit
the IFN response [5–7, 35–37]. In vitro infection with some
FCV strains does not result in activation of the IFN-bpromoter [34], thus allowing these viruses to evade the IFN
response.
Several treatment strategies against FCV disease have
been reported [30, 41]. Phosphorodiamidate morpholino
oligomers (PMO) can function as efficient drugs to control
FCV disease [30] and have been tested in natural outbreaks
of FCV. Mefloquine is a human-approved pharmaceutical
compound that has been demonstrated to be effective in the
treatment of FCV infections [18]. Combination treatment
with Mefloquine and ‘‘recombinant feline interferon x’’(rFeIFN-x) results in a higher efficiency in controlling
FCV infection [18]. However, the unfavorable aspects of
many drug metabolism pathways might restrict their
application [18]. Lithium chloride (LiCl) is widely applied
as an important therapeutic agent for nervous system dis-
orders [15], including Alzheimer’s disease [8], and for
serous ovarian cancer [20] and diabetes [12]. Several
studies have suggested that LiCl can act as an antiviral
agent for inhibiting the growth of viruses, such as coron-
avirus [27], herpes simplex virus type 1 [29], porcine
reproductive and respiratory syndrome virus (PRRSV) [11]
and infectious bronchitis virus [13]. Moreover, LiCl can
suppress host inflammatory responses [11], regulate cell
apoptosis [27], and restore the synthesis of host proteins in
virus-infected cells [43]. Our previous study found that
LiCl can efficiently suppress FCV replication in in vitro
[41]. However, antiviral therapy with a single drug often
causes the emergence of drug-resistant FCV strains. In
addition, the high evolutionary rate of FCV [4] makes it
necessary to identify more antiviral drugs.
Germacrone is one of the main constituents in volatile
oil from rhizoma curcuma [42]. It can suppress angiogen-
esis and metastasis, which contributes the restriction of cell
proliferation. Due to its potential to inhibit the growth of
tumour cells, germacrone has been widely used as an anti-
cancer drug in China [17]. It has been demonstrated that
germacrone can inhibit the replication of H1N1 and H3N2
influenza A virus in vitro by impairing the attachment/entry
step and early events in the viral life cycle [14]. Another
study also found that germacrone may be a potential drug
against porcine parvovirus (PPV) infection [3]. In the
current study, the antiviral effects of germacrone on FCV
replication were investigated by measuring changes in viral
RNA (vRNA) levels and virus yield. The results indicated
that germacrone can act as an efficient antiviral drug
against FCV replication.
Materials and methods
Viruses and cells
The FCV strains 2280, Bolin and F9 were obtained from
ATCC. Strains HRB-SS and WZ-1 were isolated from ill
household cats, as described previously [16, 41]. Crandell-
Reese feline kidney (CRFK) cells (ATCC) were cultured in
DMEM (Hyclone) supplemented with 8 % fetal bovine
serum (Gibco) and 1 % penicillin-streptomycin. Virus
stocks were propagated in CRFK cells, centrifuged at 4 �Cand 12,000 g for 20 min, and kept at -80 �C until used.
Reagents
Germacrone (CAS No. 6902-91-6) was purchased from
BELLONCAM, China. It was initially dissolved in dime-
thyl sulfoxide (DMSO; Sigma-Aldrich) at a concentration
of 20 mM in 80 % DMSO (v/v). The amount of DMSO
was constantly maintained at 0.4 % for all samples. DMSO
concentrations below 1 % (v/v) are widely used for
poorly soluble polar and nonpolar molecules and are not
toxic to retinal neuronal cell lines [9].
Cytotoxicity assay for germacrone
CRFK cells in a 96-well plate (104 cells/well) were pre-
pared prior to the assay. The next day, the culture medium
was discarded, and germacrone was added at different
concentrations (0, 10, 20, 40, 60, 80, 100 and 200 lM),
diluted in serum-free DMEM. Cells treated with DMSO
(0.4 %) were used as a control. After treatment for 24 h,
cytotoxicity assays were done using a Cell Counting Kit-8
(CCK8) (Donjindo, Japan) according to the manufacturer’s
protocol. After washing two times with 19 PBS, DMEM
(80 lL) and CCK8 solution (20 lL) were added to the
cells, and the plate was incubated at 37 �C for two hours.
The optical density (OD) was determined using an
1560 H. Wu et al.
123
EnSpire� Multimode Plate Reader (PE, USA) with a
450-nm excitation filter [41]. The relative cell viability was
calculated as a percentage of that of the mock-treated
control cells. Germacrone concentrations below the 50 %
cytostatic concentration (CC50) were considered non-toxic.
Antiviral test
To determine antiviral efficacy of germacrone against FCV
proliferation, cells were treated for one hour with germa-
crone at concentrations of 20-100 lM. After incubation,
the cells were infected with strain F9 at a multiplicity of
infection (MOI) of 0.1 TCID50 in the presence of germa-
crone for one hour. After washing three times with PBS,
the cells were exposed to germacrone (20-100 lM) for
twelve hours. The time of drug exposure in this test was
from one hour before infection until the test endpoint,
allowing the step in the viral life cycle at which the drug
acts to be determined. The virus yields in cell supernatants
were measured, total RNA was isolated from the cells, and
the levels of vRNA were determined by real-time RT-PCR.
Analysis of the effect of germacrone on viral
attachment
Cells were seeded and cultured for 24 h. Nontoxic con-
centrations (60-100 lM) of germacrone, or 0.4 % DMSO
alone for mock treatment, were mixed with the virus sus-
pension and incubated for one hour at 37 �C. The mixture
containing the drug and the virus was then added to the
cells, followed by incubation for one hour at 4 �C. TheMOI in this assay was 0.1 TCID50. The levels of vRNA in
cells were determined by real-time RT-PCR.
Analysis of the effect of germacrone on viral entry
Cells in 24-well plates were inoculated with strain F9 at an
MOI of 0.1 TCID50, which was diluted in different con-
centrations of germacrone (60-100 lM) or 0.4 % DMSO
for mock treatment for one hour at 4 �C. The plate was
then incubated for one hour at 37 �C. The levels of vRNA
were determined by real-time RT-PCR.
Effect of germacrone on viral replication
To further investigate the antiviral efficacy of germacrone,
cells were inoculated with strain F9 at an MOI of 0.1
TCID50 for one hour at 37 �C. After the cells were washedthree times with 19 PBS, the infected cells were exposed
to germacrone at concentrations of 60-100 lM or subjected
to mock treatment with 0.4 % DMSO for twelve hours. The
recovered virus yields in cell supernatants were deter-
mined. The levels of vRNA were determined by real-time
RT-PCR.
Time-of-addition assay
To determinewhich stages of the viral life cycle were affected
by the drug, the effects of simultaneous treatment, pre-treat-
ment and post-treatment were evaluated. Cells were seeded in
24-well plates and cultured for 24 hours. (1) Cells were pre-
treated with germacrone at a concentration of 60 lM for one
hour. After incubation, cells were challenged with strain F9 at
anMOI of 0.1TCID50. (2) Cellswere simultaneously exposed
to strain F9 at anMOI of 0.1 TCID50 and germacrone (60 lM)
for twelve hours. (3) Cells were inoculatedwith strain F9 at an
MOI of 0.1 TCID50 for one hour prior to exposure to 60 lMgermacrone at 1, 3, 6 and 9 hpi. The recovered virus yields in
cell supernatants were determined at 12 hpi. The levels of
vRNA were measured by real-time RT-PCR.
Evaluation of the antiviral activity of germacrone
against other FCV strains
The cells was treated with 60 lM germacrone for one hour
prior to challenge with strain 2280, Bolin, F9, HRB-SS or
WZ-1 at an MOI of 0.1 TCID50. The time of drug exposure
was from one hour before challenge until the end of the
experiment. The recovered virus yield in the cell super-
natant was determined at 12 hpi.
Virus titration
The protocol for virus titration has been described previ-
ously [35]. Briefly, virus stocks were diluted 1:10 in free-
serum DMEM and applied to the cells. After a one-hour
adsorption period, medium containing 1 % FBS was added.
At 48-72 h post-inoculation, a cytopathic effect was
observed. Following to the protocol of Reed and Muench
[26], the viral yields were calculated as the median tissue
culture infective dose log10 (TCID50/mL).
Real-time RT-PCR
The protocol for real-time RT-PCR has been described
previously [35]. Total RNA from drug-treated or untreated
cells was isolated using an RNeasy� Mini Kit (QIAGEN)
according to the manufacturer’s instructions. Transcrip-
tion of RNA into cDNA was performed using a Prime-
Script TM 1st Strand cDNA Kit (Takara, Japan). The
following primers for FCV Pro-Pol and GAPDH were
designed: FCV-for, 5’-ATGATTTGGGGTTGTGATGT-3’;
FCV-rev, 5’-TGGGGCTRTCCATGTTGAT-3’; GAPDH-
for, 5’-TGACCACAGTCCATGCCATC-3’; GAPDH-rev,
5’-GCCAGTGAGCTTCCCGTTCA-3’. The procedure for
PCR consisted of an initial step at 95 �C for 5 min, followed
by 40 cycles of 95 �C for 15 s, 55 �C for 30 s and 72 �C for
15 s. The relative level of RNA expression was determined
Inhibition of feline calicivirus by germacrone 1561
123
by the 2-DDCT method [41]. GAPDH mRNA was analyzed as
a loading control.
Indirect immunofluorescence assay (IFA)
After washing three times with PBS, cells were fixed with 4 %
paraformaldehyde for 20 min at room temperature and then
permeabilized with 0.2 % Triton X-100 for 20 min. The cells
were washed three times, and a feline anti-FCV serum (1:100,
diluted in PBS) was incubated with the cells at room temper-
ature or at 37 �C for one hour. The cells were then incubated
with FITC-conjugated goat anti-cat IgG (1:100, diluted in PBS;
Jackson, USA). The cells were then washed three times and
incubated with DAPI (1:100, diluted in PBS) at room temper-
ature for 20 min. The fluorescence was examined using an
inverted fluorescence microscope (Olympus).
Statistics
Each experiment was performed in triplicate, and data are
presented as the mean ± SD. The significance of differ-
ences was analyzed by a one-way ANOVA and an unpaired
t-test, using GraphPad Prism 5.0 software. In comparisons
between the drug- and mock-treated groups, a p-value
\0.05 was considered statistically significant and is indi-
cated as follows: **, P\ 0.01.
Results
Cytotoxicity of germacrone
Cytotoxicity assays showed that the relative cell viability
was greater than 85 % after exposure to germacrone at
concentrations ranging from 20 to 100 lM for 24 h,
whereas the viability was less than 50 % after treatment
with 200 lM germacrone for 24 h (Fig. 1). Treatment with
*100 lM germacrone (below the CC50 value) did not
result in a significant difference in cell morphology (data
not shown) compared to mock-treated cells. The concen-
tration range of 20-100 lM was therefore selected for
antiviral assays.
Identification of germacrone as an inhibitor of FCV
replication in vitro
To evaluate whether germacrone could inhibit FCV pro-
liferation, cells were treated with different concentrations
of germacrone (20-100 lM) for one hour prior to infection
with strain F9 in the presence of the drug. The results
showed that germacrone treatment (20-100 lM) led to a
significant reduction of vRNA levels compared to mock
treatment, and the inhibitory effect was dose-dependent
(Fig. 2A). Treatment with 20 lM or 40 lM germacrone
did not affect virus yield significantly, and treatment with
60-100 lM germacrone inhibited viral proliferation by at
least tenfold (Fig. 2B).
In an IFA experiment, a strong signal was observed in
mock-treated cells as well as in cells treated with 40 lMgermacrone at 12 hpi. The fluorescent signals decreased
after treatment with 60, 80 and 100 lM germacrone
(Fig. 2C). No fluorescent signals were observed in the cell
control group.
These results indicated that pre-treatment with
60-100 lM germacrone resulted in a significant reduction
in both virus titer and vRNA levels in a concentration-
dependent manner.
Germacrone treatment does not affect FCV
attachment or entry
To investigate whether the antiviral effect of germacrone is
associated with a reduction in virus attachment and entry
into cells, germacrone (60 lM-100 lM) was mixed with a
virus suspension for one hour and then added to cells, and
Fig. 1 Cell toxicity assay after germacrone treatment. Cells were
pre-exposed to 0.4 % DMSO (mock) or germacrone at concentrations
of 20, 40, 60, 80, 100 or 200 lM for 24 h. Cell toxicity was evaluated
using a CCK8 assay. The activity of DMSO-treated cells was
considered to be 100 %, and cell toxicity was plotted as the
percentage viable cells relative to mock-treated cells. The values
represent three independent experiments
cFig. 2 Evaluation of antiviral effects of germacrone on FCV growth.
(A, B) Cells were treated with germacrone at a concentration of
20-100 lM prior to challenge. After a 1-h treatment with germacrone,
cells were inoculated with strain F9 at an MOI of 0.1 TCID50 for one
hour in the presence of the drug. The time of germacrone treatment
was from one hour before challenge until the end of the experiment.
Relative vRNA levels in cells (A) and the virus yields in supernatants
from infected cells (B) were determined. (C) CRFK cells were treated
with the indicated concentrations of germacrone at 37 �C for one hour
or 0.4 % DMSO (mock) and then infected with strain F9 at an MOI of
0.1 TCID50 for one hour. Uninfected cells were used as negative
controls. Fluorescence (940) was observed at 12 hpi
1562 H. Wu et al.
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Inhibition of feline calicivirus by germacrone 1563
123
the plate was then incubated for one hour at 4 �C. Theamount of vRNA was then measured, and no difference
was found between drug-treated and mock-treated samples
(Fig. 3A), suggesting that germacrone treatment does not
affect viral attachment to cells.
To investigate whether germacrone treatment inhibits
FCV entry into cells, the cells were inoculated with virus
for one hour at 4 �C, after which the cells were exposed to
germacrone (60, 80 and 100 lM) for one hour at 37 �C.The levels of vRNA were determined by quantitative PCR.
The vRNA levels were not decreased in germacrone-trea-
ted cells (Fig. 3B), indicating that the drug did not affect
virion entry.
Germacrone treatment inhibits viral replication
Since germacrone treatment did not affect FCV attachment
and entry, we speculated that germacrone might affect viral
proliferation. Cells were infected with virus for one hour
and then treated with germacrone at a concentration of 60,
80 or 100 lM for twelve hours. The levels of vRNA and
recovered virus yields were determined.
Compared to mock-treated cells, germacrone treatment
with 60, 80 and 100 lM led to 57.51 %, 59.67 % and
69.27 % decrease, respectively, in the relative vRNA level
(Fig. 4A). Moreover, virus loads in the cells treated with
60 lM germacrone were decreased by 100-fold (Fig. 4B).
The antiviral effect of germacrone occurs chiefly
in the early stage of FCV replication
Further experiments were performed to analyze the inhi-
bitory effect of germacrone against viral proliferation.
Cells were exposed to 60 lM germacrone prior to or after
virus challenge. As shown in Fig. 5A, a reduction in the
amount of vRNA was detected at -1, 0 and 1 hpi but was
not observed from 3 h to 9 h in the germacrone-treated
groups. Compared to mock-treated cells, the virus loads
were significantly reduced in cells treated with germacrone
at -1 h, 0 h and 1 h, but no significant reduction was
detected from 3 h to 9 h (Fig. 5B). Thus, germacrone
treatment chiefly affects the early stage of viral
proliferation.
Germacrone treatment inhibits the replication
of field isolates and other reference strains
An antiviral assay was performed to evaluate whether
germacrone can affect the replication of other reference
strains and field isolates. Following a 1-h treatment with
germacrone or mock treatment, cells were infected with
different FCV isolates for one hour and treated with 60 lMgermacrone for twelve hours. The recovered virus yields
were determined at 12 hpi. As shown in Fig. 6, germacrone
treatment significantly suppressed the growth of reference
strains F9, Bolin and 2280 and field isolates WZ-1 and
HRB-SS. After germacrone treatment, a nearly tenfold
decrease in virus yield was detected for the reference
strains and field isolates compared to mock treatment
(Fig. 6).
Discussion
The family Caliciviridae is a highly diverse family of human
and non-human pathogenic viruses [28]. The human patho-
genic norovirus is a major cause of viral gastroenteritis
worldwide. For the human pathogenic norovirus, there are no
suitable in vitro culture systems, and this has restricted vac-
cine development. Antiviral drugs may be a better method to
control human norovirus infection. Feline calicivirus (FCV)
has been used widely as model systems to accelerate our
learning about HuNoVs. Developing antiviral drugs against
FCV may help in controlling human norovirus infection.
Rhizoma curcuma is applied for the treatment of tumors
and inflammation in traditional Chinese medicine [8].
Fig. 3 Evaluation of the effects of germacrone treatment on viral
attachment and entry. CRFK cells were treated as described in
‘‘Materials and methods’’, and vRNA levels in groups exposed to
germacrone or 0.4 % DMSO (mock) were analyzed during the virus
attachment stage (A) and the virus entry stage (B). The data represent
three independent experiments
1564 H. Wu et al.
123
Germacrone is a key component of the essential oils iso-
lated from rhizoma curcuma and has been used for anti-
tussive, anti-inflammatory, antidepressant, antifeedant,
antifungal, antibacterial and antitumor proposes [4, 17].
Recently, germacrone was shown to inhibit influenza virus
replication in a concentration-dependent manner [9].
PPV can cause reproductive failure in sows, and
antiviral drugs may be an alternative method to protect
animals from PPV infection. Chen et al. reported that pre-
exposure with germacrone significantly inhibited PPV
replication at an early stage in a concentration-dependent
manner [3]. Our study showed that germacrone suppressed
FCV replication as well as the production of vRNA and
progeny virus. The antiviral effect of germacrone treatment
occurred chiefly in the early stage of virus replication in a
concentration-dependent manner. The data suggest that
germacrone might be a potential drug for the treatment of
FCV disease, but further investigation is required to
examine its antiviral effect and safety in vivo. Most studies
so far have focused on in vitro data, and in vivo data are
urgently needed to explore safety issues.
Since germacrone does not affect FCV and PPV
attachment and entry, and inoculation prior to infection
results in the highest inhibitory efficiency, as in the case of
influenza virus [14], we speculate that treatment with
Fig. 4 Evaluation of the effects of germacrone treatment on viral
replication. Cells were inoculated with strain F9 at an MOI of 0.1
TCID50 for one hour. The cells were then exposed to germacrone (60-
100 lM) at 37 �C for 12 hours. Relative vRNA levels (A) in the cells
and virus yields in the cell supernatants (B) were determined
Fig. 5 The time-dependent effects of germacrone on FCV replica-
tion. Cells were inoculated with strain F9 at an MOI of 0.1 TCID50.
The cells were treated with germacrone at the indicated time points.
Relative vRNA levels (A) in the cells and virus yields in the cell
supernatants (B) were determined at 12 hpi; ‘-1 h’ indicates pre-
treatment with germacrone for one hour before virus challenge. The
data represent three independent experiments
Fig. 6 Inhibitory effects of germacrone on other reference stains and
field isolates of FCV. Cells were exposed to 60 lM germacrone for
one hour and then inoculated with the indicated FCV strains at an
MOI of 0.1 TCID50. The recovered virus yields were determined at 12
hpi. The data represent three independent experiments
Inhibition of feline calicivirus by germacrone 1565
123
germacrone may affect the expression of host genes, some
of which may be required for viral replication.
We infer that germacrone may be protective against
infection with most FCV strains. Although FCV evolves
quickly due to the low fidelity of its RNA-dependent RNA
polymerase, only one serotype has been identified so far.
Another report also demonstrated that strain F9 and seven
other strains have similar sensitivity to mefloquine treat-
ment [18]. Moreover, all of the strains used in this study,
including the currently circulating viruses from China,
showed a similar sensitivity to germacrone treatment. Our
results suggest that germacrone can be used as an effective
broad-spectrum anti-FCV drug, either alone or in combi-
nation with other anti-FCV compounds for the treatment of
cats. Additionally, the used of a combination of germa-
crone and existing antiviral drugs might help to prevent the
development of resistance to drugs developed for FCV in
the future.
Conclusions
In this study, germacrone was identified as a potent inhi-
bitor of FCV replication when present at low concentra-
tions. Germacrone treatment chiefly affected the early
phase of viral replication. Moreover, the evaluation of its
inhibitory effect against a calicivirus extends the broad
spectrum of its antiviral effects. More investigation is
needed to optimize this drug for clinical application for the
treatment of FCV. It is also necessary to analyze the
effectiveness of germacrone against other feline viruses.
Acknowledgments This study was funded by the National Natural
Science Foundation of China (No. 31402201).
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